Method for analysis using X-ray fluorescence

ABSTRACT

The present invention is a method to quantify biomarkers. The method uses an X-ray fluorescence spectrometer to perform an X-ray fluorescence analysis on the sample to obtain spectral features derived from the biomarker; and quantifying the X-ray fluorescence signal of the biomarker.

RELATED APPLICATION/CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.13/317,341 filed Oct. 14, 2011, which claims priority from provisionalapplication Ser. No. 61/455,030, filed Oct. 14, 2010, the forgoingapplications are incorporated by reference herein.

GOVERNMENT RIGHTS

This invention was made with US Government support under contract R44ES016395 awarded by the National Institute of Health. The Government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to the analysis of specimens using X-rayfluorescence.

BACKGROUND OF THE INVENTION

X-ray fluorescence (XRF) spectrometry is a powerful spectroscopictechnique that has been used to determine the elements that are presentin a sample, and to determine the quantity of those elements in thesample. The underlying physical principle of the method is that when anatom of a particular element is irradiated with X-ray radiation, theatom ejects a core electron such as a K shell electron. The resultingatom is then in an excited state, and it can return to the ground stateby replacing the ejected electron with an electron from a higher energyorbital. This is accompanied by the emission of a photon. The energy ofthe emitted photons is equal to the difference in the energies of thetwo orbitals. Each element has a characteristic set of orbital energiesand therefore, a characteristic X-ray fluorescence (XRF) spectrum.

An X-ray fluorescence spectrometer is an apparatus capable ofirradiating a sample with an X-ray beam, detecting the X-rayfluorescence from the sample, and using the X-ray fluorescence todetermine which elements are present in the sample and measuring thequantity of these elements. A typical, commercially available energydispersive X-ray fluorescence spectrometer is the EDAX Eagle XPL energydispersive X-ray fluorescence spectrometer, equipped with a microfocusX-ray tube, lithium drifted silicon solid-state detector, processingelectronics, and vendor supplied operating software, available from theEDAX division of Ametek, 91 McKee Drive Mahwah, N.J. 07430. An exampleof a wavelength dispersive X-ray fluorescence spectrometer is the ZSXPrimus, available from Rigaku Americas, 9009 New Trails Drive, TheWoodlands, Tex. 77381. In principle, any element may be detected andquantified with X-ray fluorescence.

More than 10% of cancer cases and up to 40% of emergency room casesinvolve misdiagnosis. Better analysis methods are needed. As an example,survival is associated with tumor thickness at the time of melanoma skincancer diagnosis. The World Health Organization reports that the numberof melanoma cases worldwide is increasing faster than any other cancer.An estimated 62,480 new cases and 8,420 deaths from melanoma occurred inthe US in 2008. It is estimated that one in 82 people will developmelanoma in their lifetime. Melanoma has the lowest overall survivalrate of any skin cancer and the prognosis for patients with metastasesis extremely poor, despite a variety of therapies. Metastatic melanomais highly resistant to current therapies. The risk of death frommelanoma increases as the depth of the melanoma increases. If a melanomais less than 1 millimeter deep, there is a slight chance that it hasmetastasized. Chances are greater if a melanoma has grown thicker than1.5 millimeters, i.e. a Breslow thickness of greater than 1.5 mm Earlyrecognition and correct prognosis of melanoma is extremely important inimproving survival probability, with 10-year disease-free survival ratesexceeding 90% upon early and correct diagnosis. As a second example,Autism Spectrum Disorder (ASD) and Alzheimer's Disease (AD) are thoughtto involve metal dyshomeostatis; however, adequate biomarkers do notexist for these diseases. Diagnoses are performed with questionnairesafter symptoms have already appeared, possibly well after the time whentherapies might be beneficial.

One method for estimating cancer prognosis is the use of staging-basedanalysis, which is the assessment of how much a cancer has spread.Staging systems account for tumor size, whether the tumor has invadedadjacent or distant organs, and whether metastases exist. The stage atthe time of diagnosis is a powerful predictor of survival; although,staging methods are often inadequate. In addition, treatments areselected based on the stage of a cancer. Inadequate diagnosis, staging,prognosis, and response to therapy most assuredly harm human health. Forexample, the “Breslow Depth” is a technique used for the staging andprognosis of melanoma. It may help to predict the presence of lymph nodemetastasis. However, it is subject to several sources of error.Misdiagnosis and inadequate prognostics may cause patients to receiveincorrect medications and additional invasive procedures.

Scientists are striving to find better diagnostic, prognostic, andresponse biomarkers. For example, prognostic factors that are eithercurrently used or are being assessed as melanoma diagnostics/prognosticsinclude: standard histology, immunostaining, cell type assessment andcounting, Breslow depth, Clarks level, level and depth of invasion,mitotic rate, analysis of tumor-infiltrating lymphocytes, analysis ofmicroscopic satellites. All of these face difficulties and inadequacies.

Breslow depth is measured with an ocular micrometer and requires thatthe entire tumor be excised; it is prone to inaccuracies and severalsources of error including subjective decisions by experts, variationand uniformity of staging that is not reproducible because of variationsin the depth of layers of the skin, imprecision of surgical margins, andvariability in tissue samples. Distinguishing between benign lesions andmelanomas is very difficult. An independent review by multiplepathologists is recommended, but agreement between pathologists isconsiderably variable, with disagreements in up to 40% of diagnoses asexamined by panels of experienced dermatopathologists. It isparticularly difficult to distinguish melanoma from sun-damaged skin.Specimens obtained using other biopsy techniques (e.g., punch biopsy)are even less accurate and can lead to sampling error. Diagnostic andmanagement problems arise when the initial biopsy does not sample thecomplete skin thickness or when large lesions are not sampledadequately.

Traditional histochemical staining has been used for many years fordisease diagnostics. It is performed by pathologists. Pathologistopinion of structures in stained tissue is the definitive diagnosis formost cancers and is used for prognosis, staging, therapy decisions, drugdevelopment, epidemiology, and public policy; it has been used for over150 years and no automated method has thus far proven to be acceptable.This lack of automation leads to heavy workloads for pathologists,increased costs, and numerous errors. Although histopathologicevaluation is essential for clinical investigation, it remains a timeconsuming and subjective technique, with unsatisfactory levels of inter-and intra-observer discrepancy.

Mitotic Rate has been explored as a prognostic indicator for tumors suchas melanoma in studies. However, mitotic rates vary even within tumors,and sampling can therefore be a major issue. Mitotic rate has beenlinked to tumor thickness in multivariate analysis, but remained anindependent variable in other studies. Clinical usage has not provenworthy.

Mast cell participation in immune responses, tumor progression, andvascularization has been studied in vitro. However, in situinvestigation of mast cells in routinely processed tissues is hamperedby difficulty in reliable detection of mast cells.

The use of imaging in melanoma, in particular, ultrasound, computerizedtomography, magnetic resonance imaging, and positron emission tomographyis costly and error prone. Ultrasound at 20 MHz tends to overestimatemelanoma Breslow thicknesses.

The existing state of the art for analyzing diseases or other abnormalstates is insufficient.

There remains a need for improved methods for analyzing diseases orother abnormal states.

SUMMARY OF THE PRESENT INVENTION

Briefly, the present invention includes a method to quantify biomarkers.The method includes providing a sample comprising a biomarker. Thebiomarker comprises one or more chemicals that are related to aphysiological condition. The method also includes using an X-rayfluorescence spectrometer to perform an X-ray fluorescence analysis onthe sample to obtain spectral features derived from the biomarker; andquantifying the X-ray fluorescence signal of the biomarker.

Another aspect of the present invention includes a method to diagnoseabnormal conditions. The method includes providing one or more clinicalsamples. The one or more clinical samples comprise a biomarker. Themethod further includes using an X-ray fluorescence spectrometer toperform an X-ray fluorescence analysis on at least one of the one ormore clinical samples to obtain spectral features derived from at leastone biomarker. The method further includes quantifying the X-rayfluorescence signal of the biomarker. The method optionally includes thestep of obtaining data concerning the at least one of the one or moreclinical samples using at least one other test. The method furtheroptionally includes the step of comparing the spectral features derivedfrom the biomarker to data obtained from the at least one other test.The method yet further optionally includes the step of performing adiagnosis.

Still another aspect of the present invention includes a method toidentify one or more abnormal portions of a sample. The method includesproviding a clinical sample disposed on or in a sample holder, theclinical sample comprising normal and abnormal portions. The method alsoincludes providing an X-ray fluorescence spectrometer comprising anX-ray excitation source. The method further includes using the X-rayfluorescence spectrometer to perform an X-ray fluorescence analysis onthe clinical sample to obtain spectral features derived from at leastone biomarker. The method also includes using spectral features derivedfrom the biomarker to determine whether at least one portion of thesample is normal or abnormal. The method optionally includes obtainingmultiple X-ray fluorescence analyses wherein the sample is disposed atat least two different angles relative to the excitation source. Themethod further optionally includes the step of constructing a tomogram.

Additional objects, advantages, and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic depiction of one embodiment of the presentinvention.

FIG. 2 shows a schematic depiction of another embodiment of the presentinvention.

FIG. 3 shows a schematic depiction of yet another embodiment of thepresent invention.

FIG. 4 shows an X-ray fluorescence spectrum of bovine serum albumin asdescribed in Example 1.

FIG. 5 shows an X-ray fluorescence spectrum of bovine serum albumin thatwas exposed to thimerosal, as described in Example 1.

FIG. 6 shows an overlay of an X-ray fluorescence spectrum of bovineserum albumin and an X-ray fluorescence spectrum of bovine serum albuminthat was exposed to thimerosal, as described in Example 1.

FIG. 7 shows an H&E stained tissue sample comprising regions of melanomaand regions that are amelanocytic, as described in Example 2.

FIG. 8 shows a map of the sulfur intensity of the sample shown in FIG.7, as measured by X-ray fluorescence spectrometry, as described inExample 2.

FIG. 9 shows a map of the phosphorus intensity of the sample shown inFIG. 7, as measured by X-ray fluorescence spectrometry, as described inExample 2.

FIG. 10 shows a map of the rhodium scatter intensity of the sample shownin FIG. 7, as measured by X-ray fluorescence spectrometry, as describedin Example 2.

FIG. 11 shows elemental spectrum of control cells not exposed tochromium, as described in Example 3.

FIG. 12 shows spectrum of the cells exposed to chromium for one week, asdescribed in Example 3.

FIG. 13 shows a stereoview of a biological sample as described inExample 4.

FIG. 14 shows an optical micrograph of a biological sample as describedin Example 4.

FIG. 15 shows an overlay of an X-ray fluorescence spectrum of humantransferrin with and without a 25 micron titanium filter between theX-ray source and the sample, as described in Example 5.

DETAILED DESCRIPTION

Briefly, the present invention includes a method to quantify biomarkers.The method includes providing a sample comprising a biomarker. Thebiomarker comprises one or more chemicals that are related to aphysiological condition. The method also includes using an X-rayfluorescence spectrometer to perform an X-ray fluorescence analysis onthe sample to obtain spectral features derived from the biomarker; andquantifying the X-ray fluorescence signal of the biomarker.

Another aspect of the present invention includes a method to diagnoseabnormal conditions. The method includes providing one or more clinicalsamples. The one or more clinical samples comprise a biomarker. Themethod further includes using an X-ray fluorescence spectrometer toperform an X-ray fluorescence analysis on at least one of the one ormore clinical samples to obtain spectral features derived from at leastone biomarker. The method further includes quantifying the X-rayfluorescence signal of the biomarker. The method optionally includes thestep of obtaining data concerning the at least one of the one or moreclinical samples using at least one other test. The method furtheroptionally includes the step of comparing the spectral features derivedfrom the biomarker to data obtained from the at least one other test.The method yet further optionally includes the step of performing adiagnosis.

Still another aspect of the present invention includes a method toidentify one or more abnormal portions of a sample. The method includesproviding a clinical sample disposed on or in a sample holder, theclinical sample comprising normal and abnormal portions. The method alsoincludes providing an X-ray fluorescence spectrometer comprising anX-ray excitation source. The method further includes using the X-rayfluorescence spectrometer to perform an X-ray fluorescence analysis onthe clinical sample to obtain spectral features derived from at leastone biomarker. The method also includes using spectral features derivedfrom the biomarker to determine whether at least one portion of thesample is normal or abnormal. The method optionally includes obtainingmultiple X-ray fluorescence analyses wherein the sample is disposed atat least two different angles relative to the excitation source. Themethod further optionally includes the step of constructing a tomogram.

The X-ray fluorescence analysis may be conveniently performed using anX-ray fluorescence spectrometer. An X-ray fluorescence spectrometer isan apparatus capable of irradiating a sample with an X-ray beam,detecting the X-ray fluorescence from the sample, and using the X-rayfluorescence to determine which elements are present in the sample andmeasuring the quantity of these elements. An example of an X-rayfluorescence spectrometer which may be used with the present inventionincludes the EDAX Eagle XPL energy dispersive X-ray fluorescencespectrometer, equipped with a microfocus X-ray tube, lithium driftedsilicon solid-state detector, a sample stage, processing electronics,and vendor supplied operating software, available from the EDAX divisionof Ametek, 91 McKee Drive Mahwah, N.J. 07430. An example of an X-rayfluorescence spectrometer which may be used with the present inventionincludes the ZSX Primus, available from Rigaku Americas, 9009 New TrailsDrive, The Woodlands, Tex. 77381. The X-ray fluorescence instrumentpreferably comprises at least one of the following: a monocapillaryfocusing optic, polycapillary focusing optic, a doubly curved crystalfocusing optic, a collimator, a microfocus X-ray tube, a synchrotronX-ray source, a linear accelerator X-ray source, a rhodium X-ray tube, amolybdenum X-ray tube, a chromium X-ray tube, a silver X-ray tube, apalladium X-ray tube, a monochromatic X-ray source, a polychromaticX-ray source, a polarized X-ray source, a confocal X-ray fluorescencespectrometer focusing arrangement, a PIN diode detector, a semiconductorX-ray detector, a germanium or doped germanium X-ray detector, a siliconor doped silicon X-ray detector, a wavelength dispersive X-rayfluorescence spectrometer, an energy dispersive X-ray fluorescencespectrometer, total reflectance X-ray fluorescence spectrometer, and thelike. Preferably, the X-ray excitation source emits X-ray having apolychromatic X-ray excitation spectrum, and more preferably the X-rayexcitation source emits X-rays having a spectrum with at least twomaxima. Excitation with polychromatic X-rays increases the efficiencyfor exciting more than one chemical element in the sample beinganalyzed, or for exciting more than one spectral feature in the chemicalanalyte being analyzed. The X-ray fluorescence spectrometer preferablycomprises an X-ray tube. The X-ray tube preferably consumes less than 20kilowatts of power, and more preferably consumes less than 5 kilowattsof power. The X-ray tube most preferably consumes less than five hundredwatts of power. The significance of these power levels is that benchtopequipment may be conveniently shielded against X-ray leakage when theX-ray tube has these power levels. The X-ray fluorescence spectrometerpreferably comprises one or more filters disposed between the X-rayexcitation source and the sample. The filter or filters serve toattenuate the different energy X-rays in the X-ray excitation beam todifferent degrees. Filters are useful because they can reduce unwantedX-ray signals from the sample, which often has the effect ofdramatically speeding up the X-ray fluorescence analysis. Filters areused to decrease the dead time of the X-ray fluorescence detector. Thedead time is preferably less than about 66%, and more preferably lessthan 50%, and most preferably between 0.5% and 50%. Examples of filtersinclude aluminum, titanium, iron, cellulose, chromium, nickel andrhodium, preferably having a thickness of between 2 microns and 1000microns. If the X-ray fluorescence analysis is performed on multiplespatial points on the sample, it preferable to perform the X-rayfluorescence analysis at a rate of at least one pixel per five seconds.The X-ray excitation beam preferably has a spatial cross section of lessthan about 500 microns, and more preferably less than 100 microns, forX-rays having an energy of 5,000 electron volts at the point at whichthe excitation beam impinges on the sample.

The sample is preferably measured using X-ray fluorescence spectrometryat multiple emission wavelengths, which allows multiple elements to bemeasured simultaneously. The X-ray fluorescence spectrometer performsx-ray fluorescence spectrometry. X-ray fluorescence spectrometry is usedto identify chemical elements by the energy or energies of signals inthe x-ray fluorescence spectrogram. X-ray fluorescence spectrometry isused to quantify chemical elements by the amplitude of signals, alsoreferred to as peaks, in the x-ray fluorescence spectrogram. Forexample, FIG. 4, FIG. 5, FIG. 6, FIG. 11, FIG. 12, and FIG. 15 showexamples of X-ray fluorescence spectrograms with peaks, with the peakslabeled with the chemical elements that produce the x-ray fluorescencesignals at the peaks. The area under the peaks is proportional to thequantity of the element that produces that peak, corrected for thesensitivity for that element for the x-ray fluorescence spectrometerbeing used. The area under each peak is typically derived from countingthe x-rays fluoresced or scattered from the sample having energiesassociated with the element being measured. The x-ray fluorescencespectrometer typically presents the quantity of each element measured asa spreadsheet or database with numerical data. The x-ray fluorescencespectrometer may be calibrated so that the amount of an element thatproduces a peak may be correlated to the area under that peak. It is notnecessary to generate a spectrogram and measure the area under eachpeak. X-rays may also be measured as a count rate, typically counts ofx-rays having the appropriate energies per second. The amount of x-raysat appropriate energies may also be counted. Mathematical manipulationsof the data are typically performed, for example to remove backgroundand noise and to allow statistics to be determined and to allowalgorithms to be used for diagnostic, prognostic, response, and/orhealth status biomarkers. For example, means, ratios, and/or principalcomponent analysis may be used to differentiate signals and noise andbackground. FIG. 8 shows a map constructed by plotting the intensity ofthe sulfur signals from an x-ray spectrogram obtained from a tissuesample; the contour lines and colors in FIG. 8 are derived from theintensity of the sulfur peak in each x-ray fluorescence spectrogram. Thex and y coordinates in FIG. 8 are derived from the x and y coordinatesof the measured tissue sample. FIG. 9 shows a map constructed byplotting the intensity of the phosphorus signals from an x-rayspectrogram obtained from a tissue sample; the contour lines and colorsin FIG. 9 are derived from the intensity of the phosphorus peak in eachx-ray fluorescence spectrogram. The x and y coordinates in FIG. 9 arederived from the x and y coordinates of the measured tissue sample. FIG.10 shows a map constructed by plotting the intensity of the rhodiumsignals from an x-ray spectrogram obtained from a tissue sample; thecontour lines and colors in FIG. 10 are derived from the intensity ofthe rhodium peak in each x-ray fluorescence spectrogram. The x and ycoordinates in FIG. 10 are derived from the x and y coordinates of themeasured tissue sample. Biomarkers are preferably quantified byidentifying and quantifying chemical elements in a sample that correlatewith a health condition. For example, Example 1 describes a biomarkerfor thimerosal exposure, which is quantified by the mercury x-rayfluorescence signals as well as other x-ray fluorescence signals in FIG.5 and the associated numerical data.

The sample should comprise one or more biomarkers. For the presentinvention, biomarkers are defined as portions of a sample that indicatea physiological state or process. For example, Example 1 describes abiomarker for thimerosal exposure. Example 2 describes biomarkers formelanomic and amelanomic tissue. A biomarker used with the presentinvention should preferably give rise to an x-ray fluorescence signalthat is different from the x-ray fluorescence signal of a sample thatdoes not contain the biomarker. For example, a biomarker of an abnormalstate preferably has a different empirical formula from a biomarker fora normal state. Examples of biomarkers that may be used with the presentinvention include biomarkers that allow or assist in the diagnosis,prognosis, or staging of an abnormal condition and/or assess healthstatus and/or response to therapy. Preferably the biomarker comprises achemical element having an atomic number greater than 19, and mostpreferably the biomarker is related to a disease that is associated witha different level of metal homeostatis than is found in healthypatients. Biomarkers used in the present invention may be used withadditional panels of biomarkers, such as algorithms used for cancerprognosis such as Breslow depths.

The sample is preferably deposited or mounted onto a sample holder, orinto a sample holder. Solid samples are preferably disposed on a slideor a film or a well plate. Liquid samples are preferably disposed on aslide or a film, or else may be placed in a container such as a cup or awell. The sample holder may be used to concentrate the sample toincrease X-ray fluorescence signals. The sample holder is preferablysubstantially free of at least one of the elements selected from thelist comprising sulfur, phosphorus, silicon, potassium, calcium,manganese, iron, cobalt, nickel, copper, zinc, arsenic, rhodium,molybdenum, chromium, selenium, bromine, silver, cadmium, platinum,gold, mercury, lead, gadolinium, dysprosium, terbium, europium,strontium, cesium, barium, and iodine; these elements are commonlypresent in samples to be measured, or else X-rays derived from theseelements are commonly present in measurements. Examples of suitablematerials for the deposition substrate include Porvair #229302, Porvair#229112, Porvair # 229058, Porvair # 229304, and Porvair #229301, all ofwhich are available from Porvair plc, Brampton House, 50 Bergen Way,King's Lynn, Norfolk PE30 2JG, U.K.), aluminum foils (examples:Microseal ‘F’ Foil from Bio-Rad Laboratories, 1000 Alfred Nobel Drive,Hercules, Calif. 94547). Other materials that may be used for thedeposition substrate include: polyvinylidene difluoride, polyvinylidenefluoride, cellulose, filter paper, polystyrene, agarose, Super-ThinPolyester Surface-Protection Tape, Chemical-Resistant SurlynSurface-Protection Tape, Abrasion-Resistant PolyurethaneSurface-Protection Tape, Heat-Resistant Kapton Tape with SiliconeAdhesive or with Acrylic Adhesive, UV-Resistant PolyethyleneSurface-Protection Tape, Clean-Release Polyethylene Surface-ProtectionTape, Low-Static Polyimide Tape, all available from McMaster-Carr, 6100Fulton Industrial Blvd., Atlanta, Ga. 30336-2852. Other materials whichmay be used for the deposition substrate include polypropylene,available from Lebow Company, 5960 Mandarin Ave., Goleta, Calif. 93117U.S.A. Other substrates that are conveniently used for the depositionsubstrate include AP1, AP3, ProLINE Series 10, ProLINE Series 20,DuraBeryllium substrates from Moxtek, 452 West 1260 North, Orem, Utah84057. Other materials which may be used for the deposition substrateinclude Ultralene®, mylar, polycarbonate, prolene, and kapton, availablefrom SPEX CertiPrep Ltd, 2 Dalston Gardens, Stanmore, Middlesex HA7 1BQ,ENGLAND. Other materials that may be used as the deposition substrateinclude Hostaphan®, polyester, and Etnom® available from ChemplexIndustries, Inc., 2820 SW 42nd Avenue, Palm City, Fla. 34990-5573 USA.Another material that may be conveniently used is Zone Free Film PartZAF-PE-50, available from Excel Scientific, 18350 George Blvd,Victorville, Calif., 92394. Other useful substrates are glass andsilicon. This list is not exhaustive, and other materials may be used asthe deposition substrate. The deposition substrate is also preferablysubstantially free of elements which have X-ray fluorescence emissionpeaks having energies of between 1.9 KeV and 3 KeV, because these peakstend to interfere with the signals of most interest to biochemical andbiological applications. Elements which have X-Ray Fluorescence emissionpeaks having energies of between 1.9 KeV and 3 KeV are: osmium, yttrium,iridium, phosphorus, zirconium, platinum, gold, niobium, mercury,thallium, molybdenum, sulfur, lead, bismuth, technetium, ruthenium,chlorine, rhodium, palladium, argon, silver, and thorium. If an X-rayfluorescence spectrometer is used which uses an X-ray detector whichcomprises silicon, then the deposition substrate is also preferably freeof elements which have X-ray fluorescence escape peaks (i.e. X-rayfluorescence emission peaks minus 1.74 KeV) having energies of between1.9 KeV and 3 KeV, because these escape peaks tend to interfere with thesignals of most interest for biochemical and biological applications.Elements which have X-Ray Fluorescence escape peaks having energies ofbetween 1.9 KeV and 3 KeV are: calcium, tellurium, iodine, scandium,xenon, cesium, barium, titanium, and lanthanum. “Substantially free” isdefined herein as being less than about 4% by weight. The depositionsubstrate may have additional chemical elements, which may be used formeasuring the thickness of the sample. If wavelength dispersive X-rayfluorescence is used, then the elemental purity of the depositionsubstrate is not as important; in this case, the film should besubstantially free of the element or elements which are being used toquantify the sample. The deposition substrate may be treated to increaseprotein adhesion; a non-inclusive list of treatments includes treatingthe deposition substrate with oxygen or nitrogen plasma or withpoly-lysine. The sample holder also preferably comprises at least oneelement that is not present in the sample at concentrations above oneweight percent in any pixel of the sample.

Many samples, including clinical samples, are washed or otherwiseexposed to a solution that serves to buffer the pH of the sample, bufferthe redox state of the sample, preserve the sample, fix the sample,sterilize the sample or otherwise render the sample safe or prepared forreading. Many of these solutions contain elements which might interferewith the measurement of the sample. The solution should preferably befree of at least one chemical element having an atomic number of greaterthan four, where that chemical element is present in the sample. Thesolution should more preferably be free of at least one chemical elementhaving an atomic number of greater than eight, where that chemicalelement is present in the sample. The solution should preferably be freeof at least one of the following chemicals or functional groups:dimethylsulfoxide, thiols, sulfate anion, sulfonate anions, chlorideanion, bromide anion, fluoride anion, iodide anion, perchlorate anion,phosphate anion, and phosphonate anions. The solution preferablycomprises one or more of the following chemical or functional groups:amine, imine, nitrate anion, nitrite anion, ammonium cation, acetateanion, carboxylate anion, conjugate bases of carboxylic acids, carbonateanion, formalin, formaldehyde, ethanol, 2-propanol, and iminium cation;these chemicals offer the correct chemical properties with minimal X-rayfluorescence interference. The solution is most preferably substantiallyfree of at least one element selected from the list of phosphorus andsulfur.

If the sample holder comprises at least one element which is not presentin the sample, then an X-ray fluorescence signal from the sample holdermay be compared, for example, by obtaining a ratio, to an X-rayfluorescence signal from the sample. For example, if the sample holdercomprises silicon and the sample comprises sulfur, then the ratio ofsulfur to silicon may be used to estimate the thickness of the sample.

The sample is preferably a biological or environmental specimen.Examples of biological specimens are tissue samples, such as adiposecells, adipose tissue, aneuploid regions, apoptotic regions, arthropods,bacteria, biological fluids, biopsy samples, buffy coat containingoligonucleotides, cell cycle related regions, cells, cerebrospinalfluid, differentiated regions, dried biospecimens, erythrocytes, freshbiospecimens, fungi, hemolysate, immortalized cell lines, infectiousagents, lymph nodes, lyophilized biospecimens, membranes, metastaticsites, microenvironment regions, microtome cut biospecimens, mitoticregions, nucleotides, organelles, organs, paraffin fixed biospecimens,plasma, primary cell cultures, prions, proteins, protozoa, serum, snapfrozen biospecimens, stem cells, stromal tissue, subcellularfractionates, tissue homogenate, tissue microarrays, tissues, tissuesamples showing infiltration beyond surgical margins, urine, viruses,whole blood, and the like. The sample may comprise fractionatedspecimens; for example, the sample may be one or more biologicalpolymers such as proteins or nucleic acids, amino acids, peptides,polymers comprising amino acids, oligomers comprising amino acids,nucleotides, polymers comprising nucleotides, and oligomers comprisingnucleotides. Samples may also be materials emitted or excreted from thebody, such as ear wax, urine, feces, breath, saliva, sweat, and thelike. More preferably, the sample comprises a disease related tissuesuch as a tumor specimen, such as a melanoma-containing sample.

The sample may be stained using a chemical element that is not commonlyfound in the sample, or by using a chemical comprising a chemicalelement that is not commonly found in the sample. This X-rayfluorescence staining is preferably performed using a chemical elementhaving an atomic number of 19 or greater or chemical comprising achemical element having an atomic number of 19 or greater. It isespecially convenient to use a drug that comprises one or more chemicalelements having an atomic number of 19 or greater to stain a sample. TheX-ray fluorescence analysis is quantitative, so the amount of stain andsample may be determined and a ratio may be determined Multiple assays,for example, using two different stains, where one of the stainscomprises one of the chemical elements having an atomic number of 19 orgreater and the other stain comprises a different chemical elementhaving an atomic number of 19 or greater may be performed and analyzedby X-ray fluorescence spectrometry to identify regions of the samplethat differ in chemical or biological makeup. Alternatively, multipleassays using two different stains, where one of the stains comprises oneof the chemical elements having an atomic number of 19 or greater andthe other stain does not comprise that chemical element having an atomicnumber of 19 or greater may be performed and analyzed by X-rayfluorescence spectrometry and a second analytical technique to identifyregions of the sample that differ in chemical or biological makeup.

Samples may be fractionated. If a sample is fractionated, it is oftenconvenient to homogenize the sample before fractionating. Samples may behomogenized by physical (e.g., cold denaturation or heating), mechanical(e.g., cutting, grinding, shearing, beating, shocking, sonication,blending), chemical (e.g., pH or chemical denaturation), or enzymatic(e.g., proteases) means, or a combination of these methods. The samplemay be fractionated, separated or depleted, with analysis of fractionscontaining analytes for detection of the presence, absence, or ratios oftarget analytes. Examples of fractionation techniques includecentrifugation; mechanical separation; chemical separation; enzymaticseparation; condensation; chromatographic methods such as ion exchangechromatography, affinity chromatography, immunochromatography, thinlayer chromatography, gel filtration chromatography, high performanceliquid chromatography; gel electrophoresis; isoelectric focusing; 2Delectrophoresis; precipitation; density gradient fractionation; andimmobilization of analytes to solid formats such as beads, membranes,arrays, microarrays, spin plate, or well plates before X-rayfluorescence measurements.

The sample may be analyzed by X-ray fluorescence mapping. Mappingcomprises obtaining multiple X-ray fluorescence measurements of one ormore elements, at multiple locations on the sample. The sample may bemapped while it is oriented in at least two different angles relative tothe X-ray excitation beam. The angle at which the sample is oriented tothe X-ray beam may be conveniently adjusted, for example, by tilting thestage or placing a shim underneath the sample or sample holder, or usinga sample mount capable of rotating in one or more axes. If the sample ismeasured in this manner, the X-ray fluorescence data may be displayed toappear three dimensional, for example, by projecting the images fromeach location in a different color or with imaged displayed withdifferent polarizations. The image may then viewed with glasses havingdifferent polarized lenses, different colored lenses, as cross-eye orwall-eye stereo image pairs, or using other standard three dimensionalimaging techniques. The image may also be reconstructed using computertomography to construct a three dimensional image or a tomogram.

Additional tests may be performed on the sample and the results of thesetests may be compared to the features obtained through the X-rayfluorescence analysis. Other clinical tests that may be used in thepresent invention include research or clinical methods used foridentifying, diagnosing, prognosing, staging of disease and methods usedto assess response to therapies. Examples of these methods includeanalysis using antibodies, angiogenesis scanning, biochemical assays,biomarker panels, Breslow Depth analysis, cancer staging, Clark levelanalysis, clinical exam, clinical pathology, computerized tomagraphy,darkfield miscroscopy, density analysis, electron microscopy, enzymaticor colorimetric assays, flow cytometry, fluorescence in situhybridization, genetic analysis, genotyping, hematoxylin and eosinstaining (HE staining), histological assays, magnetic resonanceangiography, immunoassays, immunohistochemistry, infrared microscopy,infrared spectroscopy, karyotyping, magnetic resonance imaging, massspectroscopy, micronutrient and macronutrient status, monochromaticX-ray fluorescence analysis, nuclear magnetic resonance spectroscopy,optical microscopy, phase contrast microscopy, phenotyping, positronemission tomography, single photon emission computed tomography,staining, synchrotron X-ray fluorescence, ultraviolet spectroscopy,ultraviolet microscopy, visible light microscopy with or without stainsand histological markers, X-ray absorbance, X-ray scatter, andfluorescence microscopy wherein fluorescent signals are intrinsic orarise from staining, and the like.

If multiple clinical samples are analyzed by X-ray fluorescence and asecond test, then preferably the clinical samples are derived from thesame patient. More preferably, the samples are obtained from at most sixinches apart from each other. Most preferably, the clinical aresufficiently proximal in space or time of collection such that at leasttwo of the clinical samples comprise the feature that is being assessed.

X-ray fluorescence data and data resulting from additional tests may beconveniently analyzed using mathematical manipulations to identifysample regions with differing compositions. Simple methods includegenerating and displaying images of the sum, difference, product orquotient of data from X-ray fluorescence and additional tests andanalyses described above. In addition, multiple data sets from X-rayfluorescence and additional tests may be analyzed together usingmultivariable statistical approaches, including component analysis,correspondence analysis, covariation analysis, dimensional reduction,factor analysis, independent component analysis, K-means clustering,neural network analyses, nonlinear dimensional reduction, principlecomponent analysis, targeted vector rotation, and the like. This list isnot exhaustive and other methods may be employed. Analyses areconveniently carried out using mathematical and statistical softwarepackages to generate statistical results and images of resultant data,including, Matlab (MathWorks, Natick Mass. 01760), IDL (ITT VisualInformation Solutions, Boulder Colo. 80301), SPSS (InternationalBusiness Machines Corp. Armonk, N.Y., 10504.) Results may be visualizedas tabulated values, or more conveniently as either two-dimensionalimages with false color or monotone color scales to represent data or asthree-dimensional surface plots displaying data as peak heights. Forcomparison of results for multiple data sources or analyses, multipledata sets may be combined using multiple colors to compare, mostconveniently using red, green and blue.

The X-ray fluorescence data may be used to diagnose a sample. The X-rayfluorescence analysis may be used to diagnose chromium exposure, asdescribed in Example 3. Regions of high phosphorus signals may be usedto diagnose aneuploidy. When a rhodium X-ray tube is use, regions ofhigh rhodium scatter signals may be used to identify adipose tissue andto diagnose obesity. Changes in ratios between multiple elements can beused to diagnose conditions such as micronutrient deficiencies anddiseases that affect metabolism of one or more specific nutrients;examples of such diseases are Wilson's disease and Menkes disease andanemia. The diagnosis is conveniently performed by comparing the X-rayfluorescence signal or signals from healthy samples and abnormal samplesto the sample being diagnosed.

The X-ray fluorescence analysis may be used to determine normal orabnormal portions of the sample, or boundaries of abnormal and normaltissue regions. The sample may be mapped using a single elemental signalin the X-ray fluorescence measurement. However, preferably, more thanone single elemental signal in the X-ray fluorescence measurement isused. Regions of high phosphorus signals, which are convenientlymeasured by the phosphorus K-alpha X-ray fluorescence peak, areassociated with post-translationally modified proteins, nucleotides, oraneuploidy; cells that undergo carcinogenesis often show abnormalnumbers of chromosomes called aneuploidy. Regions of high scattersignals, which are conveniently measured by the rhodium L-alpha X-rayfluorescence peak when using a rhodium X-ray tube for the excitationsource (as shown in Example 2), are associated with adipose tissue orfat cells; other convenient signals are the chromium K-alpha line when achromium X-ray tube is used, the molybdenum L-alpha line when amolybdenum X-ray tube is used. The density of a sample may beconveniently measured, for example, if the sample holder comprises atleast one element that is not present in the sample. In this case, theattenuation of the X-ray fluorescence signal derived from elements inthe sample holder that are not present in the sample may be correlatedto the density of the sample. Examples of normal and abnormal portionsof samples include the border between normal and abnormal, i.e. where afirst pixel shows normal tissue and the next pixel shows abnormaltissue. Other boundaries can include perinormal regions, samples thatinclude metals or other elements such as strontium or mercury,metastasized cells, dead cells, apoptotic cells, diseased cells ortissues, peritumor environments, stroma environments, diseasemicroenvironments and precursor regions. Another example of abnormalsample includes gadolinium from a magnetic resonance imaging contrastagent, such as gadopentic acid, that is not in the desired tissue. Yetanother example of abnormal sample includes platinum from aplatinum-containing chemotherapeutic agent such as cis-platin, that isnot in the tumor. Yet another example includes finding lead or mercuryin sample because this element is harmful to health and may be involvedin neurodegeneration and other diseases such as autism spectrumdisorder.

The embodiments of the present invention may be understood from thefollowing examples.

EXAMPLE 1

A solution of bovine serum albumin (150 nanomolar in pH 7.5tris(hydroxymethl)amine-hydrochloric acid buffer) was exposed tothimerosal. FIG. 4 shows the X-ray fluorescence spectra of bovine serumalbumin that was not exposed to thimerosal. FIG. 5 shows the X-rayfluorescence spectrum of bovine serum albumin that was exposed to 9millimolar thimerosal. FIG. 6 shows an expanded the X-ray fluorescencespectra of bovine serum albumin that was not exposed to thimerosal(bottom spectrum) and the X-ray fluorescence spectrum of bovine serumalbumin that was exposed to 9 millimolar thimerosal (top spectrum). Ascan be seen in FIGS. 4-6, the bovine mercury appears to displace aportion of the copper and zinc from the bovine serum albumin. Thebiomarkers for thimerosal exposure to bovine serum albumin are increasedmercury X-ray fluorescence signals, and decreased copper and zinc X-rayfluorescence signals. As an example of x-ray fluorescence data, thefollowing is a list of elements and the net counts for the spectrumshown in FIG. 5: P, 829.8 counts above background; S, 17706.6 countsabove background; Cl, 1723.2 counts above background; Rh, 38125.2 countsabove background; K, 198 counts above background; Ca, 232.8 counts abovebackground; Mn, 228 counts above background; Fe, 301.8 counts abovebackground; Co, 199.8 counts above background; Ni, 283.8 counts abovebackground; Cu, 343.8 counts above background; Zn, 349.2 counts abovebackground; Hg, 760.2 counts above background; As, 193.2 counts abovebackground; Se, 193.8 counts above background; Br, 0 counts abovebackground; Zr, 364.2 counts above background.

EXAMPLE 2

A human tissue sample comprising melanoma and amelanomic regions wasanalyzed to determine element content using X-ray fluorescence. A 12millimeter by 7.4 millimeter region was mapped with 0.1 millimeterspatial resolution using a 0.1 millimeter polycapillary X-ray focusingoptic, a rhodium X-ray tube for polychromatic X-ray excitation, andsilicon drift detector energy dispersive X-ray detector. Elementsquantified include phosphorus, sulfur, chlorine, potassium, calcium,iodine, titanium, chromium, manganese, iron, nickel, copper, zinc,platinum, mercury, arsenic, lead, selenium, cadmium, and tin. Imageswere reconstructed as three dimensional plots which represent elementconcentration in both color and peak height. FIG. 7 shows an opticalmicrograph of the H&E stained sample. FIG. 8 shows images for the sulfursignal intensity. FIG. 9 shows images for the phosphorus signalintensity. FIG. 10 shows images for the elastic scatter of the rhodiumL-line. Elastic Rh scatter correlates inversely with tissue density,while P and S signals correlate positively with tissue density. Abnormalareas are characterized by altered P to S ratios.

EXAMPLE 3

Primary white melanocytes were cultured in normal culture medium withand without chromium (VI) for one week. The cells were washed withisotonic ammonium acetate buffer. Cells were air dry and resuspended in20 mM glycine pH 2.8. Cells were then concentrated on spin plate for 2hours at 75 degrees Celsius. FIG. 11 shows elemental spectrum of controlcells not exposed to chromium, and FIG. 12 shows spectrum of the cellsexposed to Cr for one week.

EXAMPLE 4

FIG. 13 shows a 3D reconstruction of an X-ray fluorescence imageanalysis. A sample of pepperoni was analyzed to identify regions ofelement content heterogeneity. A 1 mm thick sample was mounted onpolycarbonate. A rhodium tube was used for X-ray excitation and anenergy dispersive silicon drift detector was used to simultaneouslymonitor phosphorus, sulfur, chlorine, potassium, calcium, scandium,titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper,and zinc X-ray fluorescence and rhodium elastic and inelastic scatterpeaks. Two images were acquired that differ in the angle between thesample and the X-ray excitation beam by 15 degrees. Images were producedby taking the ratio of calcium to sulfur, spatially interpolating tosmooth edges, normalizing and generating grey-scale bitmap images (FIG.13) which were simultaneously adjusted for contrast and brightness tooptimize viewing. For 3D visualization, the images are presented as across-eye stereo pair, in which the image intended for viewing by theleft eye is placed on the right, and the image intended for viewing bythe right eye is placed on the left. For correlative analysis, an imageof the same region was also acquired using standard visible lightmicroscope, as shown in FIG. 14. The stereoview X-ray fluorescenceanalysis shown in FIG. 13 identifies in three dimensions regions ofsample that differ in their elemental composition and are not observablein light microscopy. A tumor sample could have been analyzed in the samemanner.

EXAMPLE 5

A sample of dried human holo-transferrin was measured using an X-rayfluorescence spectrometer equipped with a rhodium tube for X-rayexcitation and an energy dispersive silicon drift detector tosimultaneously monitor phosphorus, sulfur, chlorine, potassium, calcium,scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel,copper, and zinc X-ray fluorescence and rhodium elastic and inelasticscatter peaks. A spectrum was obtained without a filter disposed betweenthe X-ray tube and the sample (upper spectrum in FIG. 15) and a spectrumwas obtained with a 25 micron titanium filter disposed between the X-raytube and the sample (lower spectrum in FIG. 15). The dead time withoutthe filter was 30%, and the dead time with the filter was 15%.

The foregoing description of the invention has been presented forpurposes of illustration and description and is not intended to beexhaustive or to limit the invention to the precise form disclosed, andobviously many modifications and variations are possible in light of theabove teaching.

The embodiment(s) were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed is:
 1. A method to quantify biomarkers, comprisingproviding a sample comprising a biomarker, the biomarker comprising oneor more chemicals that are related to a physiological condition;fractionating the sample to provide sample fractions; using an X-rayfluorescence spectrometer comprising an X-ray excitation source toperform an X-ray fluorescence analysis on a sample fraction to obtainspectral features derived from the biomarker; and quantifying the X-rayfluorescence signal of the biomarker; wherein the X-ray fluorescencespectrometer irradiates the sample fraction with polychromatic X-rays,and the X-ray fluorescence analysis comprises multiple X-rayfluorescence analyses obtained wherein the sample fraction is disposedat two or more different angles relative to the excitation source. 2.The method of claim 1, further comprising preparing the sample for X-rayfluorescence by exposing the sample to a solution.
 3. The method ofclaim 1, further comprising homogenizing the sample prior tofractionating the sample.
 4. The method of claim 1, whereinfractionating the sample is performed using a technique selected fromthe group of techniques consisting of centrifugation, mechanicalseparation, chemical separation, enzymatic separation, condensation,chromatographic methods, gel electrophoresis, isoelectric focusing, 2Delectrophoresis, precipitation, density gradient fractionation andimmobilization of analytes to solid formats.
 5. The method of claim 1,wherein fractionating the sample comprises immobilization of analytes tosolid formats wherein the solid formats are selected from the groupconsisting of beads, membranes, arrays, microarrays, spin plate, or wellplates.
 6. The method of claim 5, wherein fractionation comprisesimmobilization of analytes utilizing well plates.
 7. The method of claim1, wherein fractionation is performed by centrifugation.
 8. The methodof claim 1, wherein the sample fraction is disposed on or in a sampleholder, and the sample holder comprises at least one chemical elementwhich is not present in the sample.
 9. The method of claim 1, furthercomprising the step of constructing a tomogram.
 10. A method to diagnoseabnormal conditions, comprising providing one or more clinical samples,the one or more clinical samples comprising a biomarker; fractionatingthe sample to provide sample fractions; using an X-ray fluorescencespectrometer comprising an X-ray excitation source to perform an X-rayfluorescence analysis on the sample fraction to obtain spectral featuresderived from at least one biomarker; and quantifying the X-rayfluorescence signal of the biomarker, wherein the X-ray fluorescencespectrometer irradiates the sample fraction with polychromatic X-rays,and the X-ray fluorescence analysis comprises multiple X-rayfluorescence analyses obtained wherein the sample fraction is disposedat two or more different angles relative to the excitation source. 11.The method of claim 10, further comprising exposing the sample to asolution to prepare the sample for the X-ray fluorescence analysis. 12.The method of claim 10, further comprising homogenizing the sample priorto fractionating the sample.
 13. The method of claim 10, whereinfractionating the sample is performed using a technique selected fromthe group of techniques consisting of centrifugation, mechanicalseparation, chemical separation, enzymatic separation, condensation,chromatographic methods, gel electrophoresis, isoelectric focusing, 2Delectrophoresis, precipitation, density gradient fractionation andimmobilization of analytes to solid formats.
 14. The method of claim 10,wherein fractionating the sample comprises immobilization of analytes tosolid formats wherein the solid formats are selected from the groupconsisting of beads, membranes, arrays, microarrays, spin plate, or wellplates.
 15. The method of claim 14, wherein fractionation comprisesimmobilization of analytes utilizing well plates.
 16. The method ofclaim 10, wherein fractionation is performed by centrifugation.
 17. Themethod of claim 10, wherein the sample fraction is disposed on or in asample holder, and the sample holder comprises at least one chemicalelement which is not present in the sample.
 18. The method of claim 10,further comprising the step of obtaining data concerning the at leastone of the one or more clinical samples using at least one other test.19. The method of claim 18, further comprising the step of comparing thespectral features derived from the biomarker to data obtained from theat least one other test.
 20. The method of claim 10, further comprisingthe step of constructing a tomogram.
 21. A method to identify one ormore abnormal portions of a sample, comprising fractionating a clinicalsample to provide sample fractions; the clinical sample comprisingnormal and abnormal portions; disposing least one sample fraction in asample holder; providing an X-ray fluorescence spectrometer, the X-rayfluorescence spectrometer comprising an X-ray excitation source; usingthe X-ray fluorescence spectrometer to perform an X-ray fluorescenceanalysis on the sample fraction to obtain spectral features derived fromat least one biomarker; and using spectral features derived from thebiomarker to determine whether at least one portion of the samplefraction is normal or abnormal, wherein the X-ray fluorescence isperformed using excitation of the fractioned sample with polychromaticX-rays, and the X-ray fluorescence analysis comprises multiple X-rayfluorescence analyses obtained wherein the sample fraction is disposedat two or more different angles relative to the excitation source. 22.The method of claim 21, further comprising preparing the sample forX-ray fluorescence by exposing the sample to a solution.
 23. The methodof claim 21, further comprising the step of constructing a tomogram.